pGreenZeo Promoter Reporters User Manual
Contents
1. WPRE and to receive a full credit The WPRE technology is covered by patents issued to The Salk Institute for Biological Studies SBI grants you a non exclusive license to use the enclosed Product containing WPRE in its entirety for its intended use The Product containing WPRE is being transferred to you in furtherance of and reliance on such license Any use of WPRE outside of SBI s Product or the Product s intended use requires a license from the Salk Institute for Biological Studies This license agreement is effective until terminated You may terminate it at any time by destroying all Products containing WPRE in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Products containing WPRE in you control and so notify SBI in writing This License shall be governed in its interpretation and enforcement by the laws of California Contact for WPRE Licensing The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla CA 92037 888 266 5066 Toll Free 650 968 2200 outside US Page 25 System Biosciences SBI User Manual Attn Office for Technology Management Phone 858 435 4100 extension 1275 Fax 858 450 0509 CMV Promoter The CMV promoter is covered under U S Patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only An2. Volume of infecting supernatant is too high Keep the volume as low as possible to achieve maximal adsorption of viral particles to the cells 2 Transduction affects target cell viability Packaged Reporter Vector affects target cell growth Use a shorter transduction time to minimize the toxic effect to the target cells Try replacing with a similar target cell type Page 16 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA Polybrene is toxic for target cells Switch to TransDux 3 No Expression of positive control pGZ CMV reporter in target cells The CMV promoter is not functional in target cells It is a very rare case but the only way to solve this problem is to change the type of target cells V References Buchschacher G L and Wong Staal F 2000 Development of lentiviral vectors for gene theraphy for human diseases Blood 95 2499 2504 Burns J C Friedmann T Driever W Burrascano M and Yee J K 1993 Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors concentration to a very high titer and efficient gene transfer into mammalian and non mammalian cells Proc Natl Acad Sci USA 90 8033 8034 Cann A J ed 2000 RNA Viruses A Practical Approach Oxford Univ Press Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D and Naldini L 1998 A third generation lentivirus
3. and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 888 266 5066 Toll Free 650 968 2200 outside US Page 23 Vil System Biosciences SBI User Manual Licensing and Warranty Use of the pGreenZeo pRedZeo Packaged Transcriptional Reporter Lentivector i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be use
4. vector with a conditional packaging system J Virol 72 8463 8471 Gould D J and Favorov P 2003 Vectors for the treatment of autoimmune diseases Gene Therapy 10 912 927 Lee N S Dohjima T Bauer G Li H Li M J Ehsani A Salvaterra P and Rossi J 2002 Expression of small interfering 888 266 5066 Toll Free 650 968 2200 outside US Page 17 System Biosciences SBI User Manual RNAs targeted against HIV 1 rev transcripts in human cells Nature Biotechnol 20 500 505 Morgan R A Cornetta K and Anderson W F 1990 Application of the polymerase chain reaction in retroviral mediated gene transfer and the analysis of gene marked human TIL cells Hum Gene Ther 1 135 149 Pfeifer A Kessler T Yang M Baranov E Kootstra N Cheresh D A Hoffman R M and Verma I M 2001 Transduction of liver cells by lentiviral vectors Analysis in living animals by fluorescence imaging Mol Ther 3 319 322 Qin X F An D S Chen I S and Baltimore D 2003 Inhibiting HIV 1 infection in human T cells by lentiviral mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci USA 100 183 188 Quinn T P and Trevor K T 1997 Rapid quantitation of recombinant retrovirus produced by packaging cell clones Biotechniques 23 1038 1044 Sui G Soohoo C Affar E B Gay F Forrester W C and Shi Y 2002 A DNA vector based RNAi technology to suppress gene expression in ma
5. 04 11 1491 3 Curran MA Kaiser SM Achacoso PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors Mol Ther 2000 Jan 1 1 31 8 Derksen TA Sauter SL Davidson BL Feline immunodeficiency virus vectors Gene transfer to mouse retina following intravitreal injection J Gene Med 2002 Sep Oct 4 5 463 9 Haskell RE Hughes SM Chiorini JA Alisky JM Davidson BL Viral mediated delivery of the late infantile neuronal ceroid lipofuscinosis gene TPP I to the mouse central nervous system Gene Ther 2003 Jan 10 1 34 42 Price MA Case SS Carbonaro DA Yu XJ Petersen D Sabo KM Curran MA Engel BC Margarian H Abkowitz JL Nolan GP Kohn DB Crooks GM Expression from second generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells Mol Ther 2002 Nov 6 5 645 52 888 266 5066 Toll Free 650 968 2200 outside US Page 19 System Biosciences SBI User Manual Stein CS Davidson BL Gene transfer to the brain using feline immunodeficiency virus based lentivirus vectors Methods Enzymol 2002 346 433 54 Browning MT Schmidt RD Lew KA Rizvi TA Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions J Virol 2001 Jun 75 11 5129 40 Curran MA Kaiser SM Achacoso PL Nolan GP Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors Mol Ther 2000
6. Jan 1 1 31 8 Poeschla EM Wong Staal F Looney DJ Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors Nat Med 1998 Mar 4 3 354 7 Poeschla E M Looney D J and Wong Staal F 2003 Lentiviral nucleic acids and uses thereof US Patent NO 6 555 107 B2 Dull T Zufferey R Kelly M Mandel R J Nguyen M Trono D 1998 J Virol 72 8463 8471 Miyoshi H Blomer U Takashi M Gage F N Verma I M 1998 J Virol 72 8150 8157 Zufferey R Donello J E Trono D Hope T J 1999 J Virol 73 2886 2892 Ramezani A Hawley T S Hawley R G 2000 Mol Ther 2 458 469 Leung T H Hoffmann A Baltimore D 2004 Cell v 118 453 464 Page 20 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA VI Appendix A pGreenZeo pRedZeo CMV Features Feature Location fuon RSV 5 LTR 1 415 Hybrid RSV promoter RIUS long long terminal repeat required for viral packaging and transcription gags 567 919 Packaging signal nme toesson oemagna giura ansonpe onn packaging of viral transcripts Central polypurine tract includes DNA Flap cPPT 1798 1899 region involved in nuclear translocation and integration of transduced viral genome Human cytomegalovirus CMV constitutive promoter for transcription of dscGFP and zeoR Copepod green flu
7. Reporter Viruses are shipped on dry ice and should be immediately stored at 70 C upon receipt Avoid thawing and refreezing of pseudoviral particles Properly stored pseudoviral particles are stable for 6 months from the date received E Additional Required Materials e LB Agar and Broth with Ampicillin e One Shot OmniMAX2 competent cells Invitrogen Cat C8540 03 or other RecA E coli competent cells e Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 e Fetal Bovine Serum Invitrogen Cat 16000036 e Penicillin Streptomycin Invitrogen Cat 15070063 Page 10 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA e Trypsin EDTA Sigma Cat T3924 e TransDux SBI Cat LV850A 1 e Millex HV 0 45 pm PVDF filters Millipore Cat SLHVR25LS e Tissue Culture Plates and Related Tissue Culture Supplies e 293TN Human Kidney Producer Cell Line SBI Cat LV900A 1 Protocol A Key Terms MOI multiplicity of infection The ratio of infectious pseudoviral particles ifu to the number of cells being infected IFU cells MOI IFU ml infectious units per ml The relative concentration of infection competent pseudoviral particles Also called pseudoviral titer Transduction Efficiency The average copy number of expression constructs per gen
8. SBI System Biosciences pGreenZeo and pRedZeo Packaged and Plasmid Reporter Lentivectors Cat SRxxxVA PA User Manual Store kit at 80 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA Contents I Introduction and Background ececeeeeeeeeeeeeteeteeeeteeeeeeneeeeee 3 As OVGIVIOW ier aE A E EAER OAE 3 B Lentivector Differentiation Reporter and Pluripotency Monitor SysteM Sises inir iinei i iaiia iaai aia kiii 3 C pGreenZeo pRedZeo Lenti Reporter VectorsS 0068 8 D pGreenZeo pRedZeo Prepackaged Lentivirus 10 E Additional Required Materials ceeseeeeeseeeeeeeeeeeeeees 10 Ike ProtoCol ni a2 u acm an qe Ae di raa 11 Ac Key TMS oirne na a e ee eee en 11 B General COoMmMents sssesseesseeseeeresienesrnssrnssrnssrnssrrssnns 11 C pGreenZeo pRedZeo Plasmid Preparation 0 13 D Production of pGreenZeo pRedZeo packaged virus 13 E Transduction of pGreenZeo pRedZeo into target cells 14 III Example Datei ccciceiccesceciecedssieceveeeuecesevesecendetiededeedeeeteese 15 IV Troubleshooting iskra enrik Esirak En EEE EErEE EERE EE 16 Inefficient Transduction of Packaged pGZ Reporter Ve
9. al assays Due to the cell type specificity of the pGreenZeo pRedZeo reporter GFP RFP expression is only expected to occur in cell types where the particular promoter is active C pGreenZeo pRedZeo Plasmid Preparation Transformation of pGreenZeo pRedZeo lentivector reporter constructs into competent cells If you have purchased the pGreenZeo pRedZeo reporter construct as a plasmid you will need to produce enough for packaging into virus We recommend using E coli that are RecA competent cells Invitrogen One Shot OmniMAX2 Competent Cells C8540 03 Please follow the protocol according to the manufacturer s instructions The transformed E coli can be grown at 37 C on LB agar with ampicillin overnight Plasmid Purification Lentivector constructs must be grown in liquid culture LB with Ampicillin at 30 C E coli transformed with lentivector constructs seem to expel the plasmid if grown at 37 c SBI recommends the PureLink Hi Pure Plasmid Filter Purification Kit from Invitrogen Cat K2100 14 for purification of lentivector plasmids D Production of pGreenZeo pRedZeo packaged virus Please refer to the Lentivector Expression Systems Guide_to Packaging and Transduction of Target Cells manual for a full description of how to package pGreenZeo pRedZeo into VSV G pseudotyped viral particles 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual E Transduction of pG
10. ciency The design of SBs biosafe vectors has benefited researchers allowing them to conduct experimental studies with lower risk Currently SBI s vectors combine improved safety features that decrease the risk of recombination and vector mobilization with increased transduction efficiency 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Despite the above safety features use of HIV based vectors falls within NIH Biosafety Level 2 criteria due to the potential biohazard risk of possible recombination with endogenous viral sequences to form self replicating virus or the possibility of insertional mutagenesis For a description of laboratory biosafety level criteria consult the Centers for Disease Control Office of Health and Safety Web site at http www cdc gov od ohs biosfty ombl4 bmbl4s3 htm It is also important to check with the health and safety guidelines at your institution regarding the use of lentiviruses and to always follow standard microbiological practices which include e Wear gloves and a lab coat when handling the lentiviral vectors pseudoviral particles or transduced cells e Always work with pseudoviral particles in a Class Il laminar flow hood e Perform all procedures carefully to minimize splashes spills or the production of aerosols e Decontaminate work surfaces at least once a day or after any spill of viable material e Decontaminate all cultures sto
11. cks and other regulated wastes before disposal by an approved decontamination method such as autoclaving Materials to be decontaminated outside of the immediate laboratory area should be placed in a durable leakproof properly marked biohazard infectious waste container and sealed for transportation from the laboratory C pGreenZeo pRedZeo Lenti Reporter Vectors The pGreenZeo vectors express copGFP reporter gene and a zeocin selectable marker ZeoR under the control of a cell type specific promoter The WPRE element enhances the expression level of the reporter gene The pRedZeo vectors express the aRFP reporter gene instead of the copGFP reporter gene In order to facilitate the use of the lentivector based pGreenZeo system SBI offers a wide range of cell specific constructs offered as plasmid constructs or as packaged in VSV G pseudoviral particles Page 8 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat S R500VA PA SR11000VA PA RSV pUC ori pGreenZeo lenti reporter Cell specific Promoter ZeOR iza copGFP For a full list of pGreenZeo and pRedZeo reporters available please see WPRE http www systembio com stem cell research differentiation reporters catalog listings pGreenZeo pRedZeo vectors are provided as 10 ug of plasmid DNA pGreenZeo and pRedZeo lenti reporter constructs must be transduced into target cells as a packaged vi
12. ction MOI appropriate for your target cells you should do several transductions with packaged pGreenZeo pseudoviral particles at different MOI s e g from 0 1 to 5 Results of these test transductions should be used to determine an optimal MOI that yields the optimal percentage of infected cells based on the percentage of cells expressing the GFP or RFP marker Note that some cell types such as primary cells may be resistant to infection regardless of the MOI Expression of the pGreenZeo pRedZeo Reporter can be measured directly at about 48 72 hours after transduction At this time pGreenZeo pRedZeo constructs are integrated into the genomic DNA resulting in stably transduced reporter cell lines Reporter cells can be cloned in order to obtain a uniform population of the GreenZeo RedZeo cell line Some cells may express the reporter construct in 80 90 of the cells after transduction at an MOI of 1 2 For these easy to transduce cells most biological assays can be performed at 48 72 hours after transduction However some primary cells may only express the Page 12 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA construct in 10 50 of cells even when transduced at high MOP s For these difficult to transduce cells it is probably desirable to select the cells stably expressing the construct by FACS or clonal selection for experiment
13. ctor into karget Callsign e a A ETE 16 V RefCrenCe eeeccecccecceeceeneeeeneeeeeaeeeeaaeseeneeseeeesaeeesaaesseneeeaas 17 Vi APDCNCIXss i ig ieee ee ose ia teen 21 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual A pGreenZeo pRedZeo CMV Features n se 21 B Properties of the COOGFP cccccsseeeseeeeeeeetteeeeeeeeeees 22 G Related Products tiec fag te aghes ad deviheetan eect eae 23 D Technical Support 0 0 eeeeccceeeeeee eens eeeeeeeseaeeesaeeeeeeeenaees 23 VII Licensing and Warranty ccccceeeeeeeeeeeeeeeeseeeeeeeeneeees 24 Page 2 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA Introduction and Background A Overview This manual provides information describing how to use the pGreenZeo and pRedZeo Differentiation Reporter and Pluripotency Monitor Constructs and prepackaged viruses to generate stable cell lines with the constructs integrated into the host genome Before using the reagents and material supplied with this product please read the entire user manual B Lentivector Differentiation Reporter and Pluripotency Monitor Systems Eukaryotic gene expression is regulated by a wide variety of developmental and environmental stimuli First an extracellular signaling molecule binds to a specific receptor The signal is then transmitted through a s
14. d in accordance with the NIH guidelines developed for recombinant DNA and genetic research This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited without written permission by System Biosciences HIV Vector System This product is for non clinical research use only Use of this Product to produce products for resale or for any diagnostic therapeutic clinical veterinary or food purpose is prohibited In order to obtain a license to use this Product for these commercial purposes contact the Office of Research and Technology Ventures at the Dana Farber Cancer Institute Inc in Boston Massachusetts USA This Product or the use of this Product is covered by U S Patents Nos 5 665 577 and 5 981 276 and Page 24 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA foreign equivalents owned by the Dana Farber Cancer Institute Inc WPRE Technology System Biosciences SBI has a license to sell the Product containing WPRE under the terms described below Any use of the WPRE outside of SBI s Product or the Products intended use requires a license as detailed below Before using the Product containing WPRE please read the following license agreement If you do not agree to be bound by its terms contact SBI within 10 days for authorization to return the unused Product containing
15. d pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA lll Example Data The image below shows a flow cytometry measurement of GFP expression X axis in pGreenZeo CMV top row and pGreenZeo hGFAP bottom row and infected U251 GFAP positive and T98 GFAP negative malignant glioma cell lines T98 U251 GFAP negative GFAP positive CMV GFP GFAP GFP Only cells that express transcription factors that induce transcription of GFAP will fluoresce green mGFAP_GFP all cells 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual IV Troubleshooting Inefficient Transduction of Packaged pGZ Reporter Vector into Target Cells 1 Poor infection efficiency Target cells have too high or too low density Plate fewer or more cells in order to have about 50 confluency at infection stage Target cell line may be difficult to transduce Use a higher concentration less fold dilution of pseudoviral particles Optimize the transduction protocol and use HT1080 cells as a positive control cell line Polybrene added during infection stage If Polybrene is toxic to the target cells switch to TransDux Loss of pseudoviral titer during storage Ensure storage of the Packaged Reporter Vector at 70 C Each freeze thaw cycle causes reduction of the titer by 20 30 Use a fresh stock for transduction Do not keep the stock longer than 6 12 months
16. eries of molecular cascades which activate or deactivate specific transcription factors that regulate gene expression The expression of any given gene is controlled by multiple transcription factors which in turn are modulated by multiple signal transduction pathways Many of these signal transduction pathways converge at transcription factors that bind to specific transcriptional response elements found in the promoters of various genes and modulate the transcription of these genes Differentiation and pluripotency can therefore be monitored by the expression level of reporter genes controlled by promoters containing specific response elements The pGreenZeo pRedZeo reporter constructs contain promoter regions for cell specific genes that indicate pluripotency or differentiation When transcription factors present in the specific cell type bind to the cell specific promoter region the GFP or RFP and zeocin resistance reporter genes are transcribed and 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual translated into protein so that the cells fluoresce either green or red and become resistant to zeocin treatment Cell types that do not contain the specific combination of transcription factors to bind to the cell specific promoter will not have the reporter genes activated For example a cell that has differentiated into an astrocyte will contain transcription factors that bind to the GFAP gl
17. f the target cells Page 6 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA e Upon integration into the genome the 5 LTR promoter is inactivated which prevents formation of replication competent viral particles e The RSV promoter in HIV based vectors upstream of 5 LTR in the lentivector allow efficient Tat independent production of viral RNA reducing the number of genes from HIV 1 that are used in this system e The number of lentiviral genes necessary for packaging replication and transduction is reduced to three gag pol rev e The corresponding proteins are expressed from different plasmids that lack packaging signals The packaging plasmids share no significant homology to any of the expression lentivectors the pVSV G expression vector or any other vector to prevent generation of recombinant replication competent virus e None of the HIV 1 genes gag pol rev are present in the packaged viral genome as they are expressed from separate plasmids lacking packaging signal Therefore the lentiviral particles generated are replication incompetent e For pGreenZeo constructs produced pseudoviral particles will carry only a copy of your cell specific promoter sequence The choice of SBI s lentiviral system for experimental studies is driven by functional considerations including increased productivity and transduction effi
18. ial fibrillary acidic protein promoter When this combination of transcription factors binds to the GFAP promoter region in the pGreenZeo reporter vector GFP will be transcribed and the cells will fluoresce green The zeocin resistance gene will also be transcribed so that the differentiated astrocytes will be able to survive in media containing zeocin If a cell however has differentiated into a neuron it will not have the appropriate combination of transcription factors necessary to bind to the GFAP promoter The differentiated neurons will not fluoresce green and will die in the presence of zeocin 2 T2A T2A Alternatively a cell that is in a pluripotent state contains transcription factors that bind to the Oct4 promoter When this combination of transcription factors binds to the Oct4 promoter region in the pGreenZeo reporter vector GFP will be transcribed and the cells will fluoresce green The zeocin resistance gene will also be transcribed so that only the pluripotent cells are able to survive in media containing zeocin If a cell has differentiated the necessary transcription factors that bind to the Oct4 promoter are Page 4 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA no longer expressed and do not stimulate transactivation of the GFP or zeocin resistance genes The differentiated cells will not fluoresce green and will die in the p
19. indicates activation of a given transcriptional response element TRE by the cognate transcriptional factor in the natural chromosomal environment rather than in the episomal state in the nucleoplasm as is the case for conventional plasmid based TR vectors Tandem copies of integration can be avoided thus allowing for faithful promoter regulation Copy number of reporter constructs can be controlled by varying the multiplicity of infection MOI e Construction of stable reporter cell lines is possible with TR lentivectors in just several days without the need for conventional low efficiency selection of stable transfectants e Monitoring of signaling pathways by flow cytometry FACS is enabled by GFP reporters Biosafety SBI s pGreenZeo lentivectors are based on the traditional HIV vector backbone To address biosafety issues SBI uses a third generation HIV lentiviral vector Dull et al 1998 Miyoshi et al 1998 Zufferey et al 1999 Ramezani et al 2000 SBI s lentiviral vectors are efficient gene transfer vehicles as used for research applications because of their stable integration in non dividing and dividing cells and long term transgene expression SBI s HIV based lentivector systems are designed to maximize their biosafety features which include e A deletion in the enhancer of the U3 region of 3 ALTR ensures self inactivation of the lentiviral construct after transduction and integration into genomic DNA o
20. mmalian cells Proc Natl Acad Sci U S A 99 5515 5520 Curran MA Nolan GP Nonprimate lentiviral vectors Curr Top Microbiol Immunol 2002 261 75 105 Curran MA Nolan GP Recombinant feline immunodeficiency virus vectors Preparation and use Methods Mol Med 2002 69 335 50 Loewen N Barraza R Whitwam T Saenz DT Kemler Poeschla EM FIV Vectors Methods Mol Biol 2003 229 251 71 Naldini L Lentiviruses as gene transfer agents for delivery to non dividing cells Curr Opin Biotechnol 1998 Oct 9 5 457 63 Page 18 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA Sauter SL Gasmi M FIV vector systems Somat Cell Mol Genet 2001 Nov 26 1 6 99 129 Alisky JM Hughes SM Sauter SL Jolly D Dubensky TW Jr Staber PD Chiorini JA Davidson BL Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors Neuroreport 2000 Aug 21 11 12 2669 73 Brooks Al Stein CS Hughes SM Heth J McCray PM Jr Sauter SL Johnston JC Cory Slechta DA Federoff HJ Davidson BL Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus based vectors Proc Natl Acad Sci U S A 2002 Apr 30 99 9 6216 21 Crystal RG Bad for cats good for humans Modified feline immunodeficiency virus for gene therapy J Clin Invest 1999 Dec 1
21. nalysis Certificate If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2010 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 27
22. nd of the protein which shortens the half life time of the mature protein without additional transcription to 1 hour The copGFP protein is a non toxic non aggregating protein with fast protein maturation high stability at a wide range of pH pH 4 12 and does not require any additional cofactors or substrates The copGFP protein has very bright fluorescence that exceeds at least 1 3 times the brightness of EGFP the widely used Aequorea victoria GFP mutant The copGFP protein emits green fluorescence with the following characteristics emission wavelength max 502 nm excitation wavelength max 482 nm quantum yield 0 6 extinction coefficient 70 000 M cm Due to its exceptional properties copGFP is an excellent fluorescent marker which can be used instead of EGFP for monitoring delivery of lentiviral constructs into cells Page 22 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA C Related Products pPACK Lentivector Packaging Kits HIV Based pPACKH1 Packaging Kit Cat LV500A 1 FIV Based pPACKF1 Packaging Kit Cat LV100A 1 Unique lentiviral vectors that produce all the necessary lentiviral proteins and the VSV G envelope glycoprotein from vesicular stomatitis virus required to package pGreenZeo pRedZeo lentiviral constructs into pseudoviral particles D Technical Support For more information about SBI products
23. ome of target cell in the infected population B General Comments To ensure optimal results follow these general guidelines during your experiments pGZ CMV Reporter This plasmid should be used to estimate transduction efficiency of the lentiviral expression construct into target cells select the cell type with highest infection efficiency and to optimize the 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual transduction protocol Moreover the presence of copGFP positive cells indicates that the lentiviral construct can be efficiently expressed in your target cells from the CMV promoter The construct can also used for calibration of FACS machine for maximum intensity of expression pGZ mCMV Reporter Negative control construct which can be used to transduce target cells under the conditions optimized for the positive control pGZ CMV construct and determine background of GFP fluorescence of target cells with a non activated CMV promoter The transduction efficiency of the pGreenZeo pRedZeo Packaged Reporter Construct and your lentiviral expression construct may vary significantly for different cells and experimental conditions In order to optimize transduction conditions we recommend that you use HT1080 or similar cells as a positive control in parallel with your target cells and use prepackaged pGZ CMV SR501VA 1 from SBI To determine the desired multiplicity of infe
24. orescent protein similar to regular EGFP but with brighter color as a copGFP T2A zeoR 2313 3554 reporter for the transfected transduced cells a destabilizing ds peptide on the C end shortens the half life time of the mature protein to 1 hour WPRE 3565 4105 Posttranscriptional regulatory element which enhances the stability of the viral transcripts Required for viral reverse transcription self 3 ALTR AU3 4244 4477 inactivating 3 LTR with deletion in U3 region prevents formation of replication competent viral particles after integration into genomic DNA SV40 Poly A 4549 4680 Transcription termination and polyadenylation SV40 Ori 4689 4835 Allows for episomal replication of plasmid in eukaryotic cells pUC Ori 5205 5878 C Allows for high copy replication in E coli AmpR 6023 6883 C Ampicillin resistant gene for selection of the plasmid in E coli The notation C refers to the complementary strand 888 266 5066 Toll Free 650 968 2200 outside US Page 21 System Biosciences SBI User Manual B Properties of the copGFP The pGreenZeo Vectors contain the full length copGFP gene with optimized human codons for high level of expression of the fluorescent protein from the CMV promoter in mammalian cells The copGFP marker is a novel natural green monomeric GFP like protein from copepod Pontellina sp A unique feature of the copGFP protein is the presence of an additional destabilizing ds peptide on the C e
25. reenZeo pRedZeo into target cells The following protocol describes the general procedure for the transduction of the pGreenZeo pRedZeo Reporter Constructs packaged in pseudotyped viral particles into HT1080 cells This protocol assumes that you will use these guidelines in order to perform transduction of your target cells in parallel using HT 1080 cells as a positive control and can be used as a starting point for the optimization for transduction of your particular cell type Day 1 1 Plate 50 000 cells per well in a 24 well plate in cell culture medium Day 2 2 Cells should be between 50 to 70 confluent Aspirate medium from cells 3 Combine culture medium with TransDux to a 1X final concentration For example add 2 5 ul of TransDux to 500 ul culture medium and then transfer to each well 4 Add virus to each well and swirl to mix Optional Add increasing amounts of virus to different wells at varying MOls 5 10 and 20 etc to optimize the transduction Day 5 5 72 hours post transduction the viral genome will be integrated into the host cell genome Look at the cells for reporter expression if the viral construct has a reporter like GFP 6 Aspirate off medium Wash each well with PBS 7 To establish stabile cell lines you can now FACs sort for GFP or RFP positive cells If using an antibiotic selection marker you can begin your selection procedure Page 14 ver 4 071410 www systembio com pGreenZeo an
26. resence of zeocin T2A T2A Advantages of Lentivector technology Lentiviral expression vectors are the most effective vehicles for delivering genetic material to almost any mammalian cell including non dividing cells and to model organisms By packaging the lentiviral transcription reporter vector construct in pseudoviral particles you can obtain highly efficient transduction and heritable expression of transcriptional reporter constructs even with the most difficult to transfect cells like primary stem and differentiated cells In comparison to retroviral delivery systems lentivectors enter the cell nucleus without requiring cell replication Some of the advantages of lentivector technology include e Ready to use pre packaged constructs with a wide range of Transcriptional Response Elements for differentiation reporting or monitoring of pluripotency e Lentiviral reporter constructs can efficiently transduce nearly all cell types even those that are difficult to transfect such as primary or non dividing mammalian cells 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual e Our lentiviral based reporter system is a novel approach to study transcriptional regulation and offers many advantages over current transcription reporter systems TR constructs will integrate into the genome and therefore be subject to chromatin regulation Leung et al 2004 Expression of the reporter gene
27. rus in order for the constructs to function properly Transfection of the constructs into a target cell keeps the constitutive RSV promoter intact thus overriding the cell specific promoter and leading to nonspecific expression of the reporter genes 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual D pGreenZeo pRedZeo Prepackaged Lentivirus In addition to offering the pGreenZeo and pRedZeo reporter constructs as plasmids SBI also offers packaged pGreenZeo and pRedZeo reporter vectors in VSV G pseudotyped viral particles These have been produced by co transfection of the pGreenZeo pRedZeo construct and the pPACK H1 Lentiviral Packaging Plasmid Mix into 293TN producer cells Following transfection we collected the media containing the pseudoviral particles and concentrated it with PEGit then titer with the Global Ultra Rapid Titer kit For more information on SBI s virus production protocol see Lentivector Expression Systems Guide to Packaging and Transduction of Target Cells The packaged pGreenZeo pRedZeo viruses are provided as frozen pseudoviral particles The total number of infection units ifu and concentration the titer are determined using HT1080 cells and may vary for different lots of each packaged reporter vector The exact ifu titer and volume for each packaged reporter construct are indicated on its corresponding Product Analysis Certificate The Packaged Lentiviral
28. y other use of the CMV promoter requires a license from the University of lowa Research Foundation 214 Technology Innovation Center lowa City IA 52242 CopGFP Reporter This product contains a proprietary nucleic acid coding for a proprietary fluorescent protein s intended to be used for research purposes only Any use of the proprietary nucleic acids other than for research use is strictly prohibited USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN To obtain such a license please contact Evrogen_ at license evrogen com SBI has pending patent applications on various features and components of the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Page 26 ver 4 071410 www systembio com pGreenZeo and pRedZeo Packaged and Plasmid Reporter Constructs Cat SR500VA PA SR11000VA PA Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product A
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